MOHANTY, TUSHAR KUMARSINGH, LAISHRAM KIPJEN2023-11-072023-11-072020https://krishikosh.egranth.ac.in/handle/1/5810200609Estrus detection is a major problem in buffaloes due to its poor manifestation of estrus signs and higher incidences of silent estrus (29%) especially during summer. Therefore, identification of potential estrus biomarkers in easily accessible bio-fluid like saliva and cervico-vaginal mucus fluid (CVF) will be of immense use. The present study was designed to identify and validate estrus associated proteins in saliva and CVF using label free (LFQ) and labeled (TMT) method coupled with LC-MS/MS analysis. Pluriparous Murrah buffaloes (n=14) maintained at the Livestock research centre, NDRI, Karnal and a total of 18 estrous cycles were included for this study. Global proteome profiling of saliva resulted in the identification of 936, 844, 809 and 870 proteins during the proestrus, estrus, metestrus and diestrus stage of the estrous cycle. Out of these, 70, 27, 25 and 43 proteins were found specific to proestrus, estrus, metestrus and diestrus stages of estrous cycle. Differential proteome analysis of saliva using LFQ revealed the identification of 742 differentially expressed proteins (DEPs) and out of these, 133, 162 and 210 proteins were up-regulated (fold change ≥1.5) in estrus vs. proestrus, metestrus and diestrus stage. Similarly, using TMT-LC-MS/MS analysis revealed the identification of 689 DEPs in buffalo saliva and out of these, 201, 213 and 252 proteins were up regulated (fold change ≥1.5) during estrus compared to proestrus, metestrus and diestrus stage. Few vital proteins in saliva found important for estrous physiology viz., HSPA1A, VMO1, SERPINB1, SDF4, NUCB1, LCN1, OBP, and ENO3. Differential proteome analysis in CVF using LFQ-LC-MS/MS analysis identified 2463 DEPs and out of which 455, 476 proteins were up regulated during estrus compared to pre-estrus and post-estrus stage. Few important CVF proteins found upregulated during estrus phase include NUCB2, LCN1, SDF4, B2M and OBP. This study highlights the identification and validation of candidate estrus biomarker in saliva and CVF, which can be used to develop on-spot diagnostic for estrus identification, staging of the estrous cycle and deciding the exact time of insemination in buffaloes.EnglishThesis